Hematopoiesis is a physiological process where a small number of pluripotent hematopoietic stem cells (HSCs) residing in the bone marrow (BM) proliferate and differentiate to generate the full complement of blood and immune cells. Hematopoietic stem and progenitor cell (HSPC) self-renewal and lineage differentiation depend on distinct microenvironments, termed niches, defined by cellular components, soluble regulators, and the extracellular matrix. Definitive identification of the location as well as cellular and extracellular characteristics of HSPC niches in the BM has not been completed due to limitations of conventional imaging (confocal, fluorescent) techniques. In this research proposal, we employ novel imaging technologies, Laser Scanning Cytometry (LSC) and multiphoton intravital microscopy (MP-IVM), to define the localization and dynamic behavior of cell-cell interactions between HSPCs and their niche(s) in the bone marrow microenvironment. We have organized this grant proposal into two aims. Aim 1 will quantify the distribution of HSPCs in distinct anatomical locations within the extravascular BM compartment. LSC will be used to quantify and map the morphological position(s) of HSPCs in the diaphyseal (endosteal versus medullary) and metaphyseal regions of the BM. HSPCs will be identified using the bmi-1 gene targeted reporter (GFP) mouse model, where GFP levels are strongest for HSPCs, along with fluorescent immunostaining of surface antigens (c-Kit, Sca-1, flk2, CD150, CD34) characteristic of HSPC populations. LSC allows whole longitudinal sections of the femoral BM cavity to be scanned and the entire cellular content of the BM cavity (order 2.5x105 cells/image) mapped with single cell level precision, making objective quantification and statistical comparison of the localization of even extremely rare BM populations possible. Aim 2 will characterize the cellular component(s) of the HSPC niche within the BM. Multiphoton intravital microscopy (MP-IVM) will be used for in vivo analysis of the kinetics of HSPC behavior, specifically interactions between HSPCs and three distinct populations of potential HSPC niche cells: osteoblasts, vascular endothelial cells, and CXCL12-abundant reticular (CAR) cells. In vivo immunostaining will be used to identify each proposed HSPC niche cell within the BM. LSC, in conjunction with fluorescent immunostaining of surface antigens for HSPCs and niche cells, also will be used to objectively quantify the relative frequency that HSPCs are found in close association with each niche cell throughout the BM cavity. The bmi-1 targeted (GFP) reporter mouse model will again be used as the basis for identifying HSPCs. Improved definition of HSPC niche(s) in the BM may have important implications for ex vivo expansion of clinically relevant HSPC populations, and will contribute to understanding the etiology of pathological conditions such as leukemogenesis, myelodysplasia and immunodeficiency. PUBLIC HEALTH RELEVANCE: This research proposal will employ novel imaging technologies to define the localization and dynamic behavior of cell-cell interactions between hematopoietic stem and progenitor cells and their niche(s) in the bone marrow microenvironment. Such analyses should provide important insight into bone marrow environmental signals regulating hematopoietic cell development. This information may prove highly relevant to the expansion of clinically important hematopoietic stem and progenitor cell populations ex vivo, and also will contribute to understanding the etiology of pathological conditions such as leukemogenesis, myelodysplasia and immunodeficiency.